The overall object of this research has been to evaluate quinazoline analogs of folic acid (5,8-dideazafolates) as agents for the treatment of human adenocarcinoma of the large bowel. A primary basis for our approach has been that quinazolines, by acting as analogs of N5, N10-methylenetetrahydrofolate, would function as inhibitors of the folate utilizing enzymes thymidylate synthetase (TS) and dihydrofolate reductase, thus exerting an antitumor effect. In addition, by binding to TS in the presence of 5-fluoro-deoxyuridylate (FdUMP), an active metabolite of 5-fluorouracil, the quinazolines may thereby stimulate binding of FdUMP to the enzyme and possibly augment the antitumor action of 5-fluorouracil. Two quinazolines, 5,8-dideaza-folic acid (H-353) and 5,8-dideaza-10-oxa-folic acid (H-375) were purified on DEAE cellulose chromatography. The ability of the purified quinazolines H-353 and H-375 of the HCT-8 tumor in vitro was tested by conventional outgrowth techniques. The concentration of H-353 required to inhibit cell growth by 50% compared to untreated control cells (ED50) was 3.9 times 10 to the minus 6th power M after 72 hours of continuous exposure to the drug. Under the same conditions, quinazoline H-375 had an ED50 of 8.5 times 10 to the minus 7th power M. Because of its considerable activity in the HCT-8 line, H-375 will be further tested for binding to TS and dihydrofolate reductase purified from these cells, and for activity in immunologically compromised (nude) mice bearing human colon adenocarcinoma cells.